Methods for the design of molecular scaffolds and ligands

Abstract

The present invention provides methods of designing ligands that specifically bind to target molecules by testing a set of distinct compounds to identify compound that bind to member of a molecular family of interest. Those compounds that show binding activity can be used as a starting point for ligand design. Unlike previous methods, even compounds that bind with only low affinity are of interest and can be used. Common chemical structures of binding molecules including low affinity binding molecules are identified, thereby providing molecular scaffolds. Co-crystals of the protein and the molecular scaffolds can be formed and analyzed by X-ray crystallography to determine the orientation of the scaffold compounds at the binding site of the molecule. Using the orientation information on a scaffold at the binding site of the molecule, chemically tractable structures of the scaffold are identified that are modified to provide ligands that bind to the target molecule with altered binding affinity or specificity to a target molecule or to members of a molecular family. New and useful ligands can therefore be designed in a rational manner and with a minimum investment of resources using the methods provided.

What is claimed is:  
     
         1 . A method of designing a ligand that binds to at least one target molecule that is a member of a protein family, comprising 
 identifying as molecular scaffolds one or more compounds that bind to a binding site of the target molecule with low affinity;    determining the orientation of the one or more molecular scaffolds at the binding site of the target molecule by obtaining co-crystal structures of the molecular scaffolds in the binding site;    identifying one or more structures of at least one scaffold molecule that, when modified, provide a ligand having altered binding affinity or binding specificity or both for binding to the target molecule as compared to the binding of the scaffold molecule;    providing at least one said ligand.    
     
     
         2 . The method of  claim 1 , wherein said one or more molecular scaffolds bind with low affinity to a plurality of target molecules in said protein family.  
     
     
         3 . The method of  claim 1 , wherein said one more molecular scaffolds bind with very low affinity to a plurality of target molecules in said protein family.  
     
     
         4 . The method of  claim 1 , wherein providing the ligand comprises chemical synthesis.  
     
     
         5 . The method of  claim 1 , further comprising that a plurality of distinct compounds are assayed for binding to the binding site of the target molecules.  
     
     
         6 . The method of  claim 1 , further comprising that co-crystals of the molecular scaffolds bound to the target molecule are isolated, and the orientation of the molecular scaffold is determined by performing X-ray crystallography on the co-crystals.  
     
     
         7 . The method of  claim 1 , further comprising identifying common chemical structures of the molecular scaffolds and placing the molecular scaffolds into groups based on having at least one common chemical structure; and 
 determining the orientation of the one or more molecular scaffolds at the binding site of the target molecule for at least one representative compound from a plurality of groups.    
     
     
         8 . The method of  claim 1 , wherein the ligand binds to the target molecule with greater binding affinity or greater binding specificity or both than the molecular scaffold.  
     
     
         9 . The method of  claim 1 , wherein the orientation of the molecular scaffold is determined by nuclear magnetic resonance in co-crystal structure determination.  
     
     
         10 . The method of  claim 5 , wherein the plurality of distinct compounds are each assayed for binding to a plurality of members of the molecular family.  
     
     
         11 . The method of  claim 5 , wherein the distinct compounds have a molecular weight of from about 100 to about 350 daltons.  
     
     
         12 . The method of  claim 1 , wherein the target molecule is a protein and the molecular family is a protein family.  
     
     
         13 . The method of  claim 12 , wherein the protein is an enzyme.  
     
     
         14 . The method of  claim 5 , wherein the distinct compounds have a molecular weight of from about 150 to about 350 daltons.  
     
     
         15 . The method of  claim 5 , wherein the distinct compounds comprise a ring structure.  
     
     
         16 . The method of  claim 12 , wherein the protein family is selected from the group consisting of: protein kinases, proteases, and phosphatases.  
     
     
         17 . The method of  claim 7 , further comprising that after the identification of common chemical structures of the distinct compounds that bind, the compounds are grouped into classes based on common chemical structures and a representative compound from a plurality of the classes is selected for performing X-ray crystallography on co-crystals of the compound and target molecule.  
     
     
         18 . The method of  claim 14 , wherein the distinct compounds are selected based on criteria selected from the group consisting of: molecular weight, clogP, and the number of hydrogen bond donors and acceptors.  
     
     
         19 . The method of  claim 18 , wherein the clog P is less than 2, and the number of hydrogen bond donors and acceptors is less than 5.  
     
     
         20 . The method of  claim 5 , wherein the assay is an enzymatic assay.  
     
     
         21 . The method of  claim 17 , wherein the number of classes is at least 200.  
     
     
         22 . The method of  claim 1 , wherein the binding of the ligand to the target molecule causes a specific biochemical effect due to the inhibition of an enzyme.  
     
     
         23 . The method of  claim 1 , wherein the modification is the addition, subtraction, or substitution of a chemical group.  
     
     
         24 . The method of  claim 1 , wherein the modification causes the ligand to be actively transported to particular cells and/or a particular organ.  
     
     
         25 . The method of  claim 1 , wherein the modification of the compound comprises the addition or subtraction of a chemical group selected from the group consisting of: hydrogen, alkyl, alkoxy, phenoxy, alkenyl, alkynyl, phenylalkyl, hydroxyalkyl, haloalkyl, aryl, arylalkyl, alkyloxy, alkylthio, alkenylthio, phenyl, phenylalkyl, phenylalkylthio, hydroxyalkyl-thio, alkylthiocarbbamylthio, cyclohexyl, pyridyl, piperidinyl, alkylamino, amino, nitro, mercapto, cyano, hydroxyl, a halogen atom, halomethyl, an oxygen atom (forming a ketone or N-oxide) and a sulphur atom (forming a thione).  
     
     
         26 . The method of  claim 6 , further comprising that information provided by performing X-ray crystallography on the co-crystals is provided to a computer program, wherein the computer program provides a prediction of changes in the interaction between the molecular scaffold and the protein that result from specific modifications to the molecular scaffold, and the molecular scaffold is chemically modified based on the prediction of the biochemical result.  
     
     
         27 . The method of  claim 26 , wherein the computer program provides the prediction based on a virtual assay selected from the group consisting of: virtual docking of the compound to the protein, shape-based matching, free energy perturbations, and three-dimensional pharmacophore.  
     
     
         28 . The method of  claim 1 , wherein when a chemically tractable structure of the compound is modified, a ligand is provided that fills a void volume in the protein-ligand complex.  
     
     
         29 . The method of  claim 1 , wherein when a chemically tractable structure of the compound is modified, an attractive ionic charge is produced in the protein-ligand complex.  
     
     
         30 . The method of  claim 1 , further providing that the modification results in a sub-structure of the ligand being present in a binding pocket of the protein binding site when the protein-ligand complex is formed.  
     
     
         31 . The method of  claim 6 , further providing that after identifying the common chemical structures of the compounds that bind, the compounds are grouped based on comprising a common chemical sub-structure and a representative compound from a plurality of groups is selected for co-crystallization with the protein and performance of the X-ray crystallography.  
     
     
         32 . The method of  claim 6 , wherein the X-ray crystallography and the modification of a chemically tractable structure of the compound are each performed a plurality of times.  
     
     
         33 . The method of  claim 1 , wherein said molecular scaffold binds to said target molecule with very low affinity.  
     
     
         34 . The method of  claim 1 , wherein said molecular scaffold binds to said target molecule with extremely low affinity.  
     
     
         35 . The method of  claim 1 , further comprising identifying conserved residues in said binding sites that interact with said molecular scaffold.  
     
     
         36 . The method of  claim 35 , wherein identifying conserved residues comprises identifying binding site residues that are identical for a plurality of members of said molecular family in sequence alignments of said plurality of members.  
     
     
         37 . The method of  claim 36 , wherein identifying conserved residues that interact with said molecular scaffold comprises identifying conserved residues within 5 angstroms of said molecular scaffold in a co-crystal of said molecular scaffold and target molecule.  
     
     
         38 . A method of designing a ligand that binds to at least one target molecule that is a member of a molecular family, comprising, identifying as molecular scaffolds one or more compounds that bind to binding sites of a plurality of members of a molecular family; 
 determining the orientation of the one or more molecular scaffolds at the binding site of the target molecule to identify chemically tractable structures of the scaffolds that, when modified, alter the binding affinity or binding specificity between the scaffold and the target molecule;    synthesizing a ligand wherein one or more of the chemically tractable structures of the molecular scaffold is modified to provide a ligand that binds to the target molecule with altered binding affinity or binding specificity.    
     
     
         39 . The method of  claim 38 , wherein said molecular scaffold binds to said target molecule with low affinity.  
     
     
         40 . The method of  claim 38 , wherein said molecular scaffold binds to said target molecule with very low affinity.  
     
     
         41 . The method of  claim 38 , wherein said molecular scaffold binds to said target molecule with extremely low affinity.  
     
     
         42 . The method of  claim 38 , wherein said molecular scaffold binds to said target molecule with moderate affinity.  
     
     
         43 . The method of  claim 38 , further comprising that co-crystals of the molecular scaffolds bound to the target molecule are isolated, and the orientation of a molecular scaffolds at the binding site of the protein is determined by performing X-ray crystallography on the co-crystals.  
     
     
         44 . The method of  claim 38 , further comprising that common chemical structures of the molecular scaffolds are identified and that the molecular scaffolds are placed into groups based on having at least one common chemical structure; and 
 the step of determining the orientation of the one or more molecular scaffolds at the binding site of the target molecule is determined for at least one representative compound from each group.    
     
     
         45 . The method of  claim 38 , wherein the ligand binds to the target molecule with greater binding affinity or binding specificity than the molecular scaffold.  
     
     
         46 . The method of  claim 38 , wherein the orientation of the molecular scaffold is determined by nuclear magnetic resonance.  
     
     
         47 . The method of  claim 38 , wherein a plurality of distinct compounds are each assayed for binding to a plurality of members of the molecular family.  
     
     
         48 . The method of  claim 47 , wherein the distinct compounds have a molecular weight of from about 100 to about 350 daltons.  
     
     
         49 . The method of  claim 38 , wherein the target molecule is a protein and the molecular family is a protein family.  
     
     
         50 . The method of  claim 49 , wherein the protein is an enzyme.  
     
     
         51 . The method of  claim 48 , wherein the distinct compounds have a molecular weight of from about 150 to about 350 daltons.  
     
     
         52 . The method of  claim 48 , wherein the distinct compounds comprise a ring structure.  
     
     
         53 . The method of  claim 49 , wherein the protein family is selected from the group consisting of: protein kinases, proteases, and phosphatases.  
     
     
         54 . The method of  claim 47 , wherein at least about 5% of the compounds bind with low affinity.  
     
     
         55 . The method of  claim 47 , wherein at least about 10% of the compounds bind with low affinity.  
     
     
         56 . The method of  claim 44 , further comprising that after the identification of common chemical structures of the distinct compounds that bind, the compounds are grouped into classes based on common chemical structures and a representative compound from a plurality of the classes is selected for performing the X-ray crystallography.  
     
     
         57 . The method of  claim 47 , wherein the distinct compounds are selected based on criteria selected from the group consisting of: molecular weight, clogP, and the number of hydrogen bond donors and acceptors.  
     
     
         58 . The method of  claim 57 , wherein the molecular weight is from about 150 to about 350 daltons, the clog P is less than 2, and the number of hydrogen bond donors and acceptors is less than 5.  
     
     
         59 . The method of  claim 38 , wherein the assay is an enzymatic assay.  
     
     
         60 . The method of  claim 56 , wherein the number of groups is at least 200.  
     
     
         61 . The method of  claim 38 , wherein the binding of the ligand to the target molecule causes a specific biochemical effect due to the inhibition of an enzyme.  
     
     
         62 . The method of  claim 38 , wherein the modification is the addition, subtraction, or substitution of a chemical group.  
     
     
         63 . The method of  claim 38 , wherein the modification causes the scaffold to be actively transported to particular cells and/or a particular organ.  
     
     
         64 . The method of  claim 38 , wherein the modification of the compound comprises the addition or subtraction of a chemical group selected from the group consisting of: hydrogen, alkyl, alkoxy, phenoxy, alkenyl, alkynyl, phenylalkyl, hydroxyalkyl, haloalkyl, aryl, arylalkyl, alkyloxy, alkylthio, alkenylthio, phenyl, phenylalkyl, phenylalkylthio, hydroxyalkyl-thio, alkylthiocarbbamylthio, cyclohexyl, pyridyl, piperidinyl, alkylamino, amino, nitro, mercapto, cyano, hydroxyl, a halogen atom, halomethyl, an oxygen atom (forming a ketone or N-oxide) and a sulphur atom (forming a thione).  
     
     
         65 . The method of  claim 43 , further comprising that information provided by performing X-ray crystallography on the co-crystals is provided to a computer program, wherein the computer program provides a prediction of changes in the interaction between the molecular scaffold and the protein that result from specific modifications to the molecular scaffold, and the molecular scaffold is chemically modified based on the prediction of the biochemical result.  
     
     
         66 . The method of  claim 65 , wherein the computer program provides the prediction based on a virtual assay selected from the group consisting of: virtual docking of the compound to the protein, shape-based matching, free energy perturbations, and three-dimensional pharmacophore.  
     
     
         67 . The method of  claim 38 , wherein when a chemically tractable structure of the compound is modified, a ligand is provided that fills a void volume in the protein-ligand complex.  
     
     
         68 . The method of  claim 38 , wherein when a chemically tractable structure of the compound is modified, an attractive ionic charge is produced in the protein-ligand complex.  
     
     
         69 . The method of  claim 38 , further providing that the modification results in a sub-structure of the ligand being present in a binding pocket of the protein binding site when the protein-ligand complex is formed.  
     
     
         70 . The method of  claim 44 , further providing that after identifying the common chemical structures of the compounds that bind, the compounds are grouped based on comprising a common chemical sub-structure and a representative compound from each group is selected for co-crystallization with the protein and performance of the X-ray crystallography.  
     
     
         71 . The method of  claim 44 , wherein the X-ray crystallography and the modification of a chemically tractable structure of the compound are each performed a plurality of times.  
     
     
         72 . The method of  claim 38 , further comprising identifying conserved residues in said binding sites that interact with said molecular scaffold.  
     
     
         73 . The method of  claim 38 , wherein identifying conserved residues comprises identifying binding site residues that are identical for a plurality of members of said molecular family in sequence alignments of said plurality of members.  
     
     
         74 . The method of  claim 72 , wherein identifying conserved residues that interact with said molecular scaffold comprises identifying conserved residues within 5 angstroms of said molecular scaffold in a co-crystal of said molecular scaffold and target molecule.  
     
     
         75 . A method for identifying binding characteristics of a ligand of a target protein, comprising 
 identifying at least one conserved interacting residue in said target protein that interacts with at least two binding molecules; and    identifying at least one common interaction property of said at least two binding molecules with said conserved residue, thereby identifying at least one said characteristic.    
     
     
         76 . The method of  claim 75 , wherein identifying at least one conserved interacting residue comprises comparing a plurality of amino acid sequences in a protein family to which said target protein belongs and identifying binding site residues conserved in said protein family.  
     
     
         77 . The method of  claim 75 , wherein identifying at least one conserved interacting residue comprises identifying conserved residues within 5 angstroms of said at least two binding molecules in co-crystals of said binding molecules and target protein.  
     
     
         78 . The method of  claim 75 , wherein said interaction property is selected from the group consisting of hydrophobic interaction, charge-charge interaction, hydrogen bonding, charge-polar interaction, polar-polar interaction, and combinations thereof.  
     
     
         79 . A method for identifying energetically allowed sites on a binding compound for attachment of an additional component, comprising analyzing the orientation of said binding compound in a target binding site, thereby identifying accessible sites on said compound of said separate component.  
     
     
         80 . The method of  claim 79 , further comprising calculating the free energy cost of attachment of said separate component at one or more of said accessible sites.  
     
     
         81 . The method of  claim 79 , wherein said orientation is determined by co-crystallography.  
     
     
         82 . The method of  claim 79 , wherein said separate component comprises a linker.  
     
     
         83 . The method of  claim 79 , wherein said separate component comprises a label.  
     
     
         84 . The method of  claim 79 , wherein said separate component comprises a solid phase material.  
     
     
         85 . A method for attaching a binding compound to an attachment component, comprising 
 identifying energetically allowed sites for attachment of a said attachment component on a binding compound; and    attaching said compound or derivative thereof to said attachment component at said energetically allowed site.    
     
     
         86 . The method of  claim 85 , wherein said attachment component is a linker for attachment to a solid phase medium, and said method further comprises attaching said compound or derivative to a solid phase medium through said linker attached at a said energetically allowed site.  
     
     
         87 . The method of  claim 86 , wherein said linker is a traceless linker.  
     
     
         88 . The method of  claim 86 , wherein said binding compound or derivative thereof is synthesized on a said linker attached to said solid phase medium.  
     
     
         89 . The method of  claim 88 , wherein a plurality of said compounds or derivatives are synthesized in combinatorial synthesis.  
     
     
         90 . The method of  claim 86 , wherein attachment of said compound to said solid phase medium provides an affinity medium.  
     
     
         91 . The method of  claim 85 , wherein said attachment component comprises a label.  
     
     
         92 . The method of  claim 91 , wherein said label comprises a fluorophore.  
     
     
         93 . A method for making an affinity matrix, comprising 
 identifying energetically allowed sites on a target binding compound for attachment to a solid phase matrix; and    attaching said target binding compound to said solid phase matrix through a said energetically allowed site.    
     
     
         94 . The method of  claim 93 , further comprising determining the orientation of said target binding compound in a binding site in said target.  
     
     
         95 . The method of  claim 93 , wherein identifying energetically allowed sites comprises calculating the change in free energy for attachment of said target binding compound to said solid phase matrix.  
     
     
         96 . The method of  claim 93 , wherein said target binding compound is attached to said solid phase matrix through a linker.  
     
     
         97 . The method of  claim 93 , wherein said solid phase matrix is selected from the group consisting of gel, bead, plate, chip, and well.  
     
     
         98 . The method of  claim 93 , wherein identifying energetically allowed sites for attachment to a solid phase matrix is performed for at least 10 different compounds.